1.Orthopoxvirus pathogenesis A goal of EVPS is to understand virus-host interactions and exploit them for countermeasure development. Here, we sought to build on our previous experiments using a systems kinomics approach to characterize the host cell response to MPXV strains of varying virulence and to investigate similarities and differences between variola virus and MPXV to identify host targets for development of therapeutics. We are collaborating with the CDC Poxvirus and Rabies Branch (and associated WHO approval) to determine the functional host response to variola virus (VARV) insult by employing temporal host kinome analysis. Several studies have demonstrated that pharmacological targeting of cellular processes may inhibit the VARV lifecycle. Understanding how VARV modifies the host environment will allow us to both characterize the molecular host response to VARV infection and identify novel host targets for therapeutic intervention. The resultant kinome data differed significantly between the VARV and monkeypox virus (MPXV) and specific targets were verified through western blot analysis. Based on the data we have analyzed >80 kinase inhibitors for anti-poxvirus activities when added prior to, or subsequent to, viral infection. We have also performed bioinformatics analysis of VARV and MPXV data sets with focus on those derived from THP-1 monocytes and have performed: 1) Analyses for phosphorylated kinase targets that drive the differential hierarchical clustering of the VARV and MPXV data sets (top 5, 10, 25 and 50 hits) 2) Functional network analysis across all data sets to identify broader biological networks that are modulated by VARV or MPXV infection and 3) Upstream regulator network analysis to identify biological therapeutic targets for VARV and MPXV. We have also continued our studies of orthopox pathogenesis in nonhuman primates. Previously, we had established that infection of rhesus macaques with cowpox virus (CPXV) resulted in a disease that resembled human hemorrhagic smallpox, a rare and nearly 100% lethal disease associated with secondary bacterial infections. We compared the impact of antibiotic treatment on disease progression. 8 NHPs were IV inoculated with CPXV, 4 were treated with antibiotics, 4 were not. We did not observe a difference in onset of clinical signs or outcome between the groups. We did observe some differences in clinical parameters such as complete blood count and complete metabolic panel serum chemistry values. We also altered the route of inoculation of CPXV in macaques to determine if small particle aerosol results in disease that more closely resembles human smallpox. We observed an LD100 and LD50 and disease progression was different than IV inoculation and did not result in hemorrhagic signs or secondary bacterial infections. We also performed chest CT to evaluate lung progression for further development of medical imaging techniques for use in infectious disease studies. Our studies indicate the route of inoculation influences disease outcome and we plan to follow up with large particle aerosol which would alter the depth of penetration and may also alter disease progression. Our third project studying orthopox pathogenesis is based on our Backwards Matched Longitudinal Analysis which identified several cytokines that were statistically associated with lethal or non-lethal outcome in NHPs. The duration and intensity of certain cytokines was associated with survival (IFN-gamma and RANTES) or non-survival (MCP-1). The role of these cytokines was investigated using recombinant virus expressing each cytokine and knockout mice of the cytokine or its receptor. MCP-1 expressing virus and MCP-1 and MCP-1 receptor knockout mice had increased pathogenicity when compared to wild type virus. IFN-gamma expressing virus did not develop disease, and knockout of INF-gamma or its receptor resulted in increased pathogenicity. The MCP-1data suggests that macrophages are essential for controlling infection, but excessive MCP-1 alter macrophage function which may exacerbate disease. We plan follow up studies with MCP-1 inhibitors to determine if modulating host response is a viable platform for future anti-orthopox therapy. Mice inoculated with interferon gamma expressing virus did not develop any signs of disease and survived lethal challenge at the equivalent 100% lethal dose of wild type virus. We are planning to follow up to determine if expression of interferon gamma at the site of infection via a recombinant virus results in protection from challenge with the LD100 of wild type virus. 2. Bivalent vaccines that confer protection against rabies and Ebola virus We have previously developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing Zaire ebolavirus (EBOV) glycoprotein (GP) using a reverse genetics system based on the SAD B19 RABV wildlife vaccine in collaboration with Matthias Schnell of Thomas Jefferson University. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced cellular and humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. We evaluated our vaccine candidates in a rhesus macaque challenge model. 100% protection was observed with live attenuated RABV-GP. The inactivated and RVdelG-GP viruses provided 50% protection. Strong humoral and cellular immunity was observed. In summary, our findings indicated that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RVdelG-GP would appear to be an even safer alternative for use in wildlife or consideration for human use. 3.Filovirus Molecular Virology We employed temporal kinome analysis to investigate the host kinome responses to EBOV infection in human hepatocytes. Analysis of our kinome data demonstrated that transforming growth factor (TGF)-beta-mediated signaling responses were temporally modulated in response to EBOV infection. ELISA analysis of TGF-beta secretion from mock- or EBOV-infected cells demonstrated that up-regulated TGF-beta secretion correlated with the up-regulation of TGF-beta-mediated signaling. These results were further confirmed through the use of kinase inhibitors targeting cell receptors or downstream signaling pathway intermediates identified from our kinome analysis inhibited EBOV replication. In addition, kinase inhibitors targeting these signaling pathways provided a degree of protection in a lethal EBOV murine model of infection. We are also investigating the role of phosphorylation of the viral proteins in the virus lifecycle. We have chosen to focus on VP35 as it is essential for transcription and replication of the genome. Using a replicon system we have generated point mutations and clustered point mutations of potential phosphorylation sites and evaluate the mutant for replication and transcription. Our data indicates a hierarchical phosphorylation strategy that regulates VP35 function. A third project involves identification of host proteins that interact with the non-coding regions of the EBOV genome. Using Gel Mobility Shift RNase T1 protection Assays we have identified 8 regions within the Leader and Trailer elements that interact with host proteins. We are working to identify the proteins. We have identified HSC70 as interacting with at least one domain in the EBOV Trailer by pull-down assays and confirmed using IP-RT-PCR. Three HSC70 binding domains have been identified within the EBOV Trailer and mutational analysis of the 5 most region results in a decrease in replicon signal. We have also undertaken SHAPE analysis of the EBOV Trailer to define the secondary structure of the EBOV Trailer. Based on these data we may identify therapeutic targets as well as establish mechanisms of filovirus lifecycle regulation.